The most important player in the calcium induced calcium release process is the cardiac ryanodine receptor, RyR2. This is a large protein (500 kD) which forms a tetrameric channel 30x30x15 nm in size. The trans-membrane channel is believed to be formed by the C-terminal. The bulk of the molecule forms the large foot process that spans the diadic cleft between the SR and sarcolemmal membranes; its function is unknown. We plan to study the in-vivo function of this channel by a strategy of producing a null background by (tissue restricted and inducible) knockout of the wild-type gene, followed by rescue with genetically altered components. Global manipulation of the cardiac excitation-contraction coupling machinery (e.g., developing an RyR2 null myocyte in which mutated RyR2 can be expresses) produces an embryonic-lethal phenotype. The goal is to induce a spatially and temporally dependent RyR2 knock-ou in mouse heart that can be rescued by mutated forms of the human RyR2 cDNA. To avoid embryonic lethality, we are taking the following approach:Conditional and inducible gene targeting, limited to specific cardiac lineages (e.g. ventricular myocytes) and inducible at a desired developmental stage (particularly in the adult). The tools to accomplish this are the Cre recombinase - LoxP recombination system and the tetracycline trans-activator system. A mouse is constructed which carries a Cre recombinase transgene under control of a tetracycline-sensitive promoter as well as a knockout construct containing LoxP sites in such a manner that induction of Cre expression by withdrawal of tetracycline causes excision of a critical exon of the target gene. This system is placed under control of a lineage-specific promoter (such as the ventricular myosin light chain MLC2V), so that a tissue-specific knockout can be made to occur at a specified time. Currently a number of founder lines containing constructs for the MLC2v-tTA transgene, the tetop-Cre Recombinase and a reporter line have been identified and partially characterized. These lines are currently being studied for appropriate expression in a ROSA mouse background.In the case of the RyR2 knockout, a 15 kb mouse 129/SvJ genomic DNA fragment has been cloned, sequenced and confirmed to contain 4 exons of RyR2. The mutant mouse RyR2 targeting vector has been prepared in which RyR2 exons contain two flanking loxP sites and a pGK-neo resistant positive selection cassette as well as a pGK-tk negative selection cassette. The RyR2 gene targeting construct has been introduced into embryonic stem cells to establish inducible RyR2 functional channel knock-out mice to study the in vivo function of ryanodine receptor in ventricular myocardium. Chimeras have been identified and the gene has been shown to be successfully passed to the offspring. These mice are now being bred with the mice showing inducible tTA regulation in heart. - loxP, Cre recombinase, ryanodine receptor, EC coupling, mice, heart